金松双黄酮抗骨质疏松症作用的机制研究
发布时间：2018-10-24 21:31
【摘要】：骨是一种动态变化的组织。破骨细胞的骨吸收活性和成骨细胞的骨形成活性,共同维持骨的动态平衡。这种平衡被打破容易导致骨疾病的发生。破骨细胞的数量过多或是骨吸收活性过强,将会导致骨质流失,容易引起骨质疏松症。目前骨质疏松的治疗方案主要是通过降低骨吸收或促进骨形成来实现的。有报道称金松双黄酮(Sc)具有保护成骨细胞,促进骨形成的作用,但是对破骨细胞的影响和作用机制尚未有报道。本研究旨在探讨金松双黄酮对破骨细胞的影响及其作用机制,以及对LPS(脂多糖)诱导的小鼠骨丢失模型的影响。本研究通过体外分离培养小鼠骨髓单核细胞(BMMs),并用细胞因子M-CSF和RANKL诱导分化成破骨细胞。用不同浓度的金松双黄酮溶液刺激破骨细胞,探讨对破骨细胞的生存、分化和功能的影响。采用Real-time PCR和Westernblotting方法检测金松双黄酮对破骨细胞相关基因的转录以及对NF-κB和MAPK信号通路的影响。结果显示金松双黄酮能够抑制破骨细胞的分化成熟和骨吸收活性,并有剂量依赖性。同时还能够抑制破骨细胞相关基因,如Cts K、MMP-9、TRAP、NFATc1和C-Fos的转录,抑制NF-κB信号通路的活化,但是对MAPK信号通路的磷酸化并无作用。为了进一步研究金松双黄酮对骨质疏松症的治疗效果,我们使用LPS腹腔注射建立了小鼠骨丢失模型。LPS是革兰氏阴性菌外壁的重要组成部分,促进炎性因子的产生,如TNF-α、IL-6、IL-1β等,造成体内炎性环境,促进破骨细胞的形成和活化,导致炎性骨丢失。在本研究中,小鼠被随机分为对照组(SHAM)、模型组(LPS)和加药组(LPS+Sc)。小鼠每隔两天注射一次7.5μg/g体重量的LPS或盐水,在LPS注射前1h,给予药物或盐水。LPS首次注射后第八天安乐死处死小鼠,收集小鼠腿骨。采用TRAP染色、HE染色和Micro-CT方法,检测金松双黄酮对破骨细胞的活性以及骨组织结构的影响。结果显示,金松双黄酮在体内能够抑制破骨细胞的形成,明显改善由LPS引起的骨小梁断裂、骨密度降低。通过以上实验得出,在体外,金松双黄酮通过抑制NF-κB信号通路活化,下调下游破骨细胞分化关键转录因子C-Fos和NFATc1的表达水平,进而抑制破骨细胞分化标志性基因的表达,最终使得破骨细胞的分化成熟和骨吸收功能得到抑制。在体内,金松双黄酮同样可以抑制破骨细胞的活性,改善由LPS引起的骨丢失症状。体内和体外实验结果表明,金松双黄酮具有抑制破骨细胞分化及骨吸收功能的作用,可以作为治疗骨质疏松症的候选药物进一步研究。
[Abstract]:Bone is a kind of dynamic tissue. The bone resorption activity of osteoclasts and the osteogenic activity of osteoblasts together maintain the dynamic balance of bone. This balance is broken and can easily lead to bone disease. Excessive number of osteoclasts or excessive bone resorption may lead to bone loss and osteoporosis. The current treatment of osteoporosis is mainly achieved by reducing bone resorption or promoting bone formation. It has been reported that Jin Song bisflavone (Sc) can protect osteoblasts and promote bone formation, but the effect of Jin Song on osteoclasts and its mechanism have not been reported. The purpose of this study was to investigate the effect of Jin Song bisflavone on osteoclasts and its mechanism, and on the bone loss model induced by LPS (lipopolysaccharide) in mice. In this study, mouse bone marrow monocytes (BMMs),) were isolated and cultured in vitro and induced to differentiate into osteoclasts by cytokines M-CSF and RANKL. The effects of different concentrations of Jin Song bisflavone solution on the survival, differentiation and function of osteoclasts were studied. Real-time PCR and Westernblotting were used to detect the effect of Jin Song bisflavone on the transcription of osteoclast related genes and on NF- 魏 B and MAPK signaling pathway. The results showed that Jin Song could inhibit osteoclast differentiation, maturation and bone resorption in a dose-dependent manner. It also inhibited the transcription of osteoclast related genes, such as Cts, MMP-9, TRAPP, NFATc1 and C-Fos, and inhibited the activation of NF- 魏 B signaling pathway, but had no effect on the phosphorylation of MAPK signaling pathway. In order to further study the therapeutic effect of Jin Song bisflavone on osteoporosis, we established a model of bone loss in mice by intraperitoneal injection of LPS. LPS is an important component of the outer wall of Gram-negative bacteria and promotes the production of inflammatory factors, such as TNF- 伪, IL-6,IL-1 尾, etc. Cause inflammatory environment, promote osteoclast formation and activation, leading to inflammatory bone loss. In this study, mice were randomly divided into control group (SHAM), model group (LPS) and admixture group (LPS Sc). Mice were injected with 7.5 渭 g / g LPS or saline every two days, and then given drugs or saline one hour before LPS injection. The mice were euthanized on the eighth day after the first LPS injection, and the leg bones of the mice were collected. The effects of Jin Song bisflavone on osteoclast activity and bone structure were detected by TRAP staining, HE staining and Micro-CT staining. The results showed that Jin Song could inhibit the formation of osteoclasts in vivo, improve the trabecular fracture induced by LPS, and decrease bone mineral density (BMD). From the above experiments, it can be concluded that in vitro, Jin Song bisflavone can inhibit the activation of NF- 魏 B signaling pathway and down-regulate the expression of C-Fos and NFATc1, the key transcription factors of downstream osteoclast differentiation, and then inhibit the expression of the signature genes of osteoclast differentiation. Finally, osteoclast differentiation, maturation and bone resorption were inhibited. In vivo, Jin Song bisflavone also inhibited the activity of osteoclasts and improved the symptoms of bone loss caused by LPS. The results of in vivo and in vitro experiments showed that Jin Song bisflavone could inhibit osteoclast differentiation and bone resorption and could be used as a candidate drug for the treatment of osteoporosis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580
[Abstract]:Bone is a kind of dynamic tissue. The bone resorption activity of osteoclasts and the osteogenic activity of osteoblasts together maintain the dynamic balance of bone. This balance is broken and can easily lead to bone disease. Excessive number of osteoclasts or excessive bone resorption may lead to bone loss and osteoporosis. The current treatment of osteoporosis is mainly achieved by reducing bone resorption or promoting bone formation. It has been reported that Jin Song bisflavone (Sc) can protect osteoblasts and promote bone formation, but the effect of Jin Song on osteoclasts and its mechanism have not been reported. The purpose of this study was to investigate the effect of Jin Song bisflavone on osteoclasts and its mechanism, and on the bone loss model induced by LPS (lipopolysaccharide) in mice. In this study, mouse bone marrow monocytes (BMMs),) were isolated and cultured in vitro and induced to differentiate into osteoclasts by cytokines M-CSF and RANKL. The effects of different concentrations of Jin Song bisflavone solution on the survival, differentiation and function of osteoclasts were studied. Real-time PCR and Westernblotting were used to detect the effect of Jin Song bisflavone on the transcription of osteoclast related genes and on NF- 魏 B and MAPK signaling pathway. The results showed that Jin Song could inhibit osteoclast differentiation, maturation and bone resorption in a dose-dependent manner. It also inhibited the transcription of osteoclast related genes, such as Cts, MMP-9, TRAPP, NFATc1 and C-Fos, and inhibited the activation of NF- 魏 B signaling pathway, but had no effect on the phosphorylation of MAPK signaling pathway. In order to further study the therapeutic effect of Jin Song bisflavone on osteoporosis, we established a model of bone loss in mice by intraperitoneal injection of LPS. LPS is an important component of the outer wall of Gram-negative bacteria and promotes the production of inflammatory factors, such as TNF- 伪, IL-6,IL-1 尾, etc. Cause inflammatory environment, promote osteoclast formation and activation, leading to inflammatory bone loss. In this study, mice were randomly divided into control group (SHAM), model group (LPS) and admixture group (LPS Sc). Mice were injected with 7.5 渭 g / g LPS or saline every two days, and then given drugs or saline one hour before LPS injection. The mice were euthanized on the eighth day after the first LPS injection, and the leg bones of the mice were collected. The effects of Jin Song bisflavone on osteoclast activity and bone structure were detected by TRAP staining, HE staining and Micro-CT staining. The results showed that Jin Song could inhibit the formation of osteoclasts in vivo, improve the trabecular fracture induced by LPS, and decrease bone mineral density (BMD). From the above experiments, it can be concluded that in vitro, Jin Song bisflavone can inhibit the activation of NF- 魏 B signaling pathway and down-regulate the expression of C-Fos and NFATc1, the key transcription factors of downstream osteoclast differentiation, and then inhibit the expression of the signature genes of osteoclast differentiation. Finally, osteoclast differentiation, maturation and bone resorption were inhibited. In vivo, Jin Song bisflavone also inhibited the activity of osteoclasts and improved the symptoms of bone loss caused by LPS. The results of in vivo and in vitro experiments showed that Jin Song bisflavone could inhibit osteoclast differentiation and bone resorption and could be used as a candidate drug for the treatment of osteoporosis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580
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